A filter paper dye-binding assay for quantitative determination of protein without interference from reducing agents or detergents.
A method is described for quantitation of protein in the presence of reducing agents, detergents, and other substances which often interfere with assays of protein in solution. The proteins are applied to Whatman No. 1 filter paper, air-dried, washed with methanol, and then stained with Coomassie brilliant blue G. Following destaining, the paper is air-dried and the protein-bound dye is extracted. Sample absorbance measurements are made in a 96-well plate using an automated microplate reader (600-405 nm) or in a cuvette at 610 nm. This filter paper assay is useful for determining 100 ng to 20 micrograms of protein in the presence of ammonium sulfate, urea, thiol-reducing agents, amino acids, DNA, ionic and nonionic detergents, and acid or base.
Minamide
L S
LS
Department of Biochemistry, Colorado State University, Fort Collins 80523.
Bamburg
J R
JR
D000818:Animals; D004396:Coloring Agents / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D003902:Detergents / Q000378:metabolism*; D004229:Dithiothreitol / Q000378:metabolism; D005374:Filtration / Q000295:instrumentation; D005998:Glycine / Q000378:metabolism; D008297:Male; D000432:Methanol; D008832:Microchemistry / Q000379:methods; D010047:Ovalbumin / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D012044:Regression Analysis; D012474:Salmon; D014238:Trichloroacetic Acid
D004396:Coloring Agents; D003902:Detergents; D011506:Proteins; D014238:Trichloroacetic Acid; D010047:Ovalbumin; D004247:DNA; D004229:Dithiothreitol; D005998:Glycine; D000432:Methanol